Method of treating lupus nephritis

ABSTRACT

Methods of treating patients and evaluating patients for disease stage and/or severity are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 11/953,360,filed Dec. 10, 2007, now U.S. Pat. No. 8,048,635, issued Nov. 1, 2011,which is a continuation application claiming priority under 35 U.S.C.§120 of International Application No. PCT/US2006/022830, filed on Jun.12, 2006, and claims the benefit of U.S. provisional Application No.60/689,905, filed on Jun. 13, 2005, all of which are hereby incorporatedby reference in their entirety.

BACKGROUND

The identification of genes and proteins linked with the severity and/orprogression of disease and the development of diagnostic methods toidentify and/or monitor disease progression are of considerableimportance.

SUMMARY OF THE INVENTION

The invention is based, at least in part, on the discovery that Tweakand/or Tweak receptor, e.g., Fn14 (and certain proteins that aremodulated, e.g., induced, by Tweak) can be used as biomarkers of diseaseactivity in a biological fluid of a human subject. Accordingly, methodsand compositions are provided for assessing, staging, and/or monitoringdisease activity in a subject, e.g., assessing, staging, and/ormonitoring inflammatory disease activity (e.g., lupus, fibrosis,rheumatoid arthritis, multiple sclerosis, and nephritis activity, e.g.,lupus nephritis); and neurodegenerative disease activity.

Accordingly, in one aspect, the invention features a method ofevaluating a subject. The method includes evaluating Tweak or Fn14(and/or certain proteins that are increased by Tweak), in a biologicalfluid of a subject (such as a human), and correlating the result of theevaluation with the subject's risk, stage or status of disease activity,e.g., inflammatory disease activity. The subject can have, or be at riskfor, e.g., an inflammatory condition such as lupus, rheumatoid arthritis(RA), psoriatic arthritis (PsA), multiple sclerosis, nephritis (e.g.,interstitial nephritis, lupus nephritis, glomerulonephritis (GN),mesangial GN, membraneous GN, diffuse proliferative GN and/ormembranoproliferative GN), stroke or a neurodegenerative disease (e.g.,ALS, Parkinson's Disease, Huntington's Disease, Alzheimer's Disease),fibrosis, or cancer (e.g., solid cancers and/or hematological cancers).The term “correlating” means describing the relationship between thepresence or level of the protein or nucleic acid, and the stage, status,extent, severity, or level of risk for disease. Such correlation may bedisplayed in a record, e.g., a print or computer readable material,e.g., an informational, diagnostic, or instructional material. Therecord may identify the presence or level of a Tweak or Tweak-R proteinor nucleic acid as a diagnostic, staging or prognostic factor for thedisease. The record may include a parameter (qualitative orquantitative) representing expression or activity of Tweak and/orTweakR, as evaluated by the method.

The evaluation is performed on a biological fluid from the subject,e.g., serum, urine, plasma, cerebrospinal fluid (CSF), or synovialfluid. Increased Tweak or TweakR protein levels in the fluid correlate(e.g., directly) with increased severity, stage and/or activity ofdisease. The ability to perform such an evaluation on a readilyobtainable biological fluid from a subject provides a simple, quick,relatively non-invasive method for evaluating, staging, and/ordiagnosing a subject, e.g., before, during and/or after a treatment isbegun.

In one embodiment, increased Tweak or TweakR urinary, serum or plasmalevels correlate with increased severity or activity of renal disease(e.g., more advanced stage or increased severity of nephritis, e.g.,lupus nephritis) compared to a reference value. In another embodiment,increased Tweak or TweakR serum, plasma, urine or synovial fluid levelscorrelate with increased severity or activity of RA compared to areference value. In another embodiment, increased Tweak or TweakR serum,plasma, or urine fluid levels correlate with increased severity oractivity of lupus compared to a reference value. In another embodiment,increased Tweak or TweakR serum, plasma, urine or CSF levels correlatewith increased severity or activity of MS or a neurodegenerative diseasecompared to a reference value. In another embodiment, increased Tweak orTweakR serum, plasma, or urine levels correlate with increased severityor activity of fibrosis compared to a reference value. A reference valuecan be a control value, e.g., a value for a normal subject (e.g., asubject not suspected of, or at risk for, the disease being evaluated),a value determined for a cohort of subjects, or a baseline (e.g., prior)value from the subject being evaluated.

The method can be used to stage and/or diagnose a disorder, e.g., todiagnose the stage or severity of the disorder; to evaluate thesubject's response to treatment, e.g., to monitor progression orimprovement in a parameter of the disorder in a subject being treatedfor the disorder; to evaluate the course of the disorder, e.g., toassess the risk of, or to predict a flare-up of, the disorder. In oneembodiment, the evaluation is performed more than once, e.g., atperiodic intervals over a period of time, e.g., to monitor progressionof the disease or to monitor response to a treatment. For example, theevaluation may be performed daily, every 2 or 3 days, every week, every2 weeks, monthly, every 6 weeks, every 2 months, every 3 months or asappropriate, over a period of time to encompass at least 2, 3, 5, 10evaluations or more.

In some embodiments, the step of evaluating includes detectingexpression or activity of a Tweak or Tweak-R protein or a nucleic acidencoding Tweak or Tweak-R (e.g., by qualitative or quantitative analysisof mRNA, cDNA, or protein), or evaluating one or more nucleotides in anucleic acid (genomic, mRNA, or cDNA) encoding Tweak or Tweak-R. In oneembodiment, the method includes using an immunoassay to detect Tweakprotein, e.g., in a biological fluid, such as a urine sample, of thesubject. In other embodiments, the method can include administering alabeled Tweak or Tweak-R binding agent (e.g., an antibody) to a subject,and evaluating localization of the labeled binding agent in the subject,e.g., by imaging the subject (e.g., imaging at least a portion of thekidney of the subject).

In one embodiment, the subject has nephritis, or is suspected of havingnephritis. The method can be used to evaluate a treatment for renaldisease, e.g., nephritis, e.g., lupus nephritis. For example, thesubject is receiving a treatment for renal disease and the subject isevaluated before, during, or after receiving the treatment, e.g.,multiple times during the course of treatment. The subject may havenormal kidney function as defined and detected by a clinical measure,e.g., urine protein level, blood creatinine level, urine creatininelevel, creatinine clearance, and/or blood urea nitrogen. In other cases,the subject has an abnormal, e.g., deficient, kidney function, e.g., asdefined and detected by a clinical measure.

In one embodiment, the subject has arthritis, e.g., rheumatoid arthritis(RA) or psoriatic arthritis (PsA), or is suspected of having arthritis.The method can be used to evaluate a treatment for arthritis. Forexample, the subject is receiving a treatment for arthritis (e.g., ananti-TNF therapy, methotrexate or steroids) and the subject is evaluatedbefore, during, or after receiving the treatment, e.g., multiple timesduring the course of treatment.

In one embodiment, the subject has lupus. The subject may have, e.g.,lupus nephritis, and/or neuropsychiatric manifestations (CNS lupus) orother manifestations of lupus, e.g., low blood count, serositis orhematological manifestations. The method can be used to evaluate atreatment for lupus. For example, the subject is receiving a treatmentfor lupus (e.g., NSAID, corticosteroids, or a DMARD) and the subject isevaluated before, during, or after receiving the treatment, e.g.,multiple times during the course of treatment.

In one embodiment, the subject has a neurodegenerative disease, e.g.,MS, Parkinson's Disease, Huntington's Disease, Alzheimer's Disease orALS, or is suspected of having a neurodegenerative disease. The methodcan be used to evaluate a treatment for a neurodegenerative disease. Forexample, the subject is receiving a treatment for a neurodegenerativedisease (e.g., beta-interferon, riluzole, a cholinesterase inhibitor,copaxone, an NMDA receptor antagonist) and the subject is evaluatedbefore, during, or after receiving the treatment, e.g., multiple timesduring the course of treatment.

The method can be used to identify a subject for treatment, e.g., fortreatment for nephritis, lupus, fibrosis, rheumatoid arthritis,psoriatic arthritis, stroke, cancer or a neurodegenerative disease. Thesubject can be identified as a subject suited for treatment as afunction of results of the detection, e.g., the results show similarityto, e.g., statistically significant similarity to, a reference valueindicative of a subject being at a particular stage of the particulardisease. For example, elevated Tweak or Tweak-R expression in the urine,serum, plasma, or CSF can be indicative of a subject who can be treatedwith a Tweak or Tweak-R blocking agent or other treatment for thedisease.

An increase identified by the evaluation step, e.g., a statisticallysignificant increase, or at least 20%, 25%, 30, 50, 70, 80, 100, or 110%increase, in a subject, over a reference value, e.g., a base value orcontrol, can indicate increased activity or severity of the disease,e.g., lupus nephritis (e.g., membranous, focal proliferative, or diffuseproliferative lupus nephritis). For example, a 10%, 20%, 25% or greaterincrease in urine Tweak levels (e.g., relative to creatinine) comparedto a negative control, baseline value, or previous evaluation in asubject indicates increased nephritis severity or activity. In oneembodiment, Tweak levels greater than 0.5, 1, 2, 3, 4, or 5 pg/mg Cr canindicate that the subject has nephritis, e.g., lupus nephritis. In oneembodiment, Tweak levels in the range of 2-8 pg/mg Cr (e.g., about 2,2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, or 7.5 pg/mg Cr) can indicatethat the subject has stable disease; Tweak levels in the range of 8-10pg/mg Cr can indicate that the subject is at risk for a relapse; Tweaklevels greater than 10 pg/mg Cr (e.g., between 10-15 pg/mg Cr) canindicate that the subject is in active relapse.

In one embodiment, the method includes evaluating the subject (e.g.,evaluating urine samples from the subject) a plurality of instances overtime, e.g., over the course of a treatment, e.g., at least one day, fivedays, a week, four or six weeks, and so forth. The method can includedetermining a rate of change for the evaluated parameter, e.g., todetermine disease progression or therapeutic efficacy. In oneembodiment, the subject is also being treated, e.g., with a Tweakblocking agent or other treatment for the disease.

In one embodiment, the evaluation includes contacting a biologicalsample of the subject, preferably a urine, serum, plasma, CSF orsynovial fluid sample, with an agent that detects Tweak, TweakR or abiomarker whose expression is modulated (e.g., increased) by Tweak(e.g., in mesangial cells). The agent, e.g., an antibody or nucleic acidprobe, can be immobilized on a solid phase, e.g., on a microtiter well,tube, dipstick or other test device. In a preferred embodiment, theexpression, presence, level, or activity is detected using a dipstick orother test device format assay.

The evaluation can include a protein-based (e.g., an immunoassay) ornucleic acid-based assay, e.g., a hybridization-based assay. In oneembodiment, the evaluating step comprises performing one or more of:enzyme-linked immunoassay, radioimmunoassay, immunoblot assay (includingWestern blot analysis and sandwich assay), in situ hybridization,Northern blot analysis, and nucleic acid amplification, including PCR(e.g. quantitative RT-PCR). Many evaluation methods can include one ormore features of the foregoing. Exemplary immunoassays can includecontacting the sample with an antibody that binds to Tweak or can beadapted to use other agents that bind to Tweak, e.g., a soluble Tweakreceptor. Nucleic acid-based assays can include hybridization with anucleic acid from a Tweak-encoding sequence, e.g., from a humanTweak-encoding genomic sequence or cDNA, e.g., the coding or non-codingstrand, or a primer or other oligonucleotide complementary to a regionof a Tweak-encoding sequence. It is also possible to evaluate abiomarker that is modulated (e.g., increased) by Tweak.

In some embodiments, evaluation can be facilitated by a dipstick orother test device-based kit, e.g., suitable for testing by non-trainedindividuals, e.g., suitable for home testing. Such a screening testwould provide convenience, privacy and eliminate the necessity and costof visiting a physician for a screening test, although the dipstick orother test device kit could also be used in a clinical setting. Thedipstick or other test device kit could be similar to a home pregnancykit, known to those of skill in the art, and could provide a colorindication for an increased risk, stage or severity for an inflammatorycondition, e.g., nephritis, based upon the levels of a protein describedherein, e.g., Tweak, TweakR or a biomarker whose expression modulated(e.g., increased) by Tweak, in the sample. Such a dipstick or other testdevice-based kit could be provided with a small plastic cup forcollecting and retaining the sample and for conducting the test. In onescenario, the dipstick or other test device can react to produce onecolor if a reference level of a first protein, e.g., Tweak, is exceeded,a different color if a reference level of a second protein, e.g., abiomarker whose expression modulated (e.g., increased) by Tweak, isexceeded, and when both levels are exceeded, the two colors will combineto yield a third color that is easily distinguishable from the others.For example, a dipstick or other test device that turns yellow when areference level of Tweak is exceeded, and turns blue when a referencelevel of the biomarker is exceeded will turn green when both levels areexceeded. Because a dipstick or other test device-based assay kit wouldbe relatively resistant to temperature and humidity variations, it couldeasily be transported, stored and used virtually anywhere. In oneembodiment, the kit includes at least 1, e.g., at least 2, 5, 10, 20,30, or 50, test devices, e.g., dipsticks, e.g., membranes, e.g.,membrane strips described herein. In one embodiment, the kit contains acontainer suitable for collecting a urine sample. The kit can alsocontain a device to obtain a tissue sample, such as a cotton swab orwooden swab.

In another embodiment, the method of the present invention may beutilized in combination with a densitometer in a device for use in asetting such as a doctor's office, a clinic or a hospital. Thedensitometer can provide rapid measurement of the optical density ofdipstick or other test device strips that have been contacted with abodily fluid or tissue.

In one embodiment, the method additionally includes treating the subjectfor the condition being evaluated, e.g., nephritis. For example, themethod includes: identifying a subject at risk for or having lupusnephritis. The method can further include providing the subject atreatment for lupus, e.g., a Tweak blocking agent or other treatmentsuitable for treating lupus nephritis, e.g., corticosteroids or otherimmunosuppressive medications.

In a preferred embodiment, the subject is further evaluated for one ormore of the following parameters: albumin levels; glucose levels; urinecreatine; urine protein, and so forth.

In a preferred embodiment, the evaluation is used to choose a course oftreatment. For example, if the subject is determined to be at risk forloss of renal function or renal disease, the treatment can includedietary restrictions.

In one embodiment, the evaluation is performed by the subject. Inanother embodiment, the evaluation is performed by a health careprovider. In yet another embodiment, the evaluation is performed by athird party.

The method can also be used to select a patient population fortreatment. A set of one or more subjects indicated for renalinflammation, e.g., relative to a reference are selected. The subjectsof the set are administered a Tweak blocking agent or other treatmentfor renal inflammation, e.g., for renal nephritis.

Subjects can also be evaluated in response to other indications, e.g.,signs of early disease, e.g., loss of renal function, when a renaldisorder, e.g., early loss of renal function, is diagnosed; before,during or after a treatment for an a renal disorder, is begun or beginsto exert its effects; or generally, as is needed to maintain health,e.g., kidney health, e.g., throughout the natural aging process. Theperiod over which the agent is administered (or the period over whichclinically effective levels are maintained in the subject) can be longterm, e.g., for six months or more or a year or more, or short term,e.g., for less than a year, six months, one month, two weeks or less.

The method can also include obtaining a profile, e.g., the profileincluding parameters (qualitative or quantitative) representingexpression or activity of a plurality of biomarkers, e.g., one or moreof (preferably at least two of): Tweak and a biomarker whose expressionmodulated (e.g., increased) by Tweak (e.g., RANTES, KC, and/or IP-10).The profile can be compared to a reference profile, e.g., usingmulti-dimensional analysis, e.g., distance functions. A computer mediumcan be used that has executable code for effecting one or more thefollowing steps: receive a subject expression profile; access a databaseof reference expression profiles; and either i) select a matchingreference profile most similar to the subject expression profile or ii)determine at least one comparison score for the similarity of thesubject expression profile to at least one reference profile. Thesubject expression profile, and the reference expression profiles eachinclude a value representing the level of expression of Tweak RANTES,KC, and/or IP-10. The record can further include a subject identifier,e.g., a patient identifier, and optionally other clinical information,e.g., information that assesses a inflammatory response or autoimmuneresponse.

Targeting of the Tweak pathway (e.g., with a Tweak pathway inhibitorsuch as an agent that blocks a Tweak-TweakR interaction, e.g., ananti-Tweak or anti-Fn14 blocking antibody) can be used in the treatmentof nephritis, e.g., lupus nephritis.

In another aspect, the disclosure features a method of treatingnephritis. The method includes: administering, to a subject (e.g., ahuman subject) who has or is at risk for nephritis, a Tweak blockingagent, e.g., in an amount and for a time to provide a therapeuticeffect. In one embodiment, the agent is an antibody, e.g., a Tweak orFn14 antibody, or a soluble form of Tweak receptor, e.g., a solubleFn14.

In one embodiment, the subject has lupus. In such embodiments, the Tweakblocking agent is effective to treat the renal manifestations of thelupus, e.g., to treat lupus nephritis.

In one embodiment, the disorder is acute nephritis, chronic nephritis,glomerulonephritis (GN), primary glomerulonephritis, autoimmunenephritis, pyelonephritis, mesangial GN, membraneous GN, diffuseproliferative GN membranoproliferative GN and/or interstitial nephritis.In one embodiment the nephritis is not rapidly progressive crescenticglomerulonephritis.

In one embodiment, the agent is an antibody that is a full length IgG.In other embodiments, the agent is an antigen-binding fragment of a fulllength IgG, e.g., the agent is a single chain antibody, Fab fragment,F(ab′)2 fragment, Fd fragment, Fv fragment, or dAb fragment. Inpreferred embodiments, the antibody is a human, humanized or humaneeredantibody or antigen-binding fragment thereof.

In one embodiment, the agent is a soluble form of the Tweak receptor,e.g., a polypeptide at least 95% identical to amino acids 28-X1 of SEQID NO:2, where amino acid X1 is selected from the group of residues 68to 80 of SEQ ID NO:2. In some cases, the soluble form of the receptor isfused with a heterologous polypeptide, e.g., an antibody Fc region.

In one embodiment, the agent is administered in an amount sufficient toreduce urinary protein levels, delay or prevent additional kidneyfunction deterioration, and/or improve kidney function.

In one embodiment, the agent is administered at a dose between 0.1-100mg/kg, between 0.1-10 mg/kg, between 1 mg/kg-100 mg/kg, between 0.5-20mg/kg, or 1-10 mg/kg. In the most typical embodiment, the dose isadministered more than once, e.g., at periodic intervals over a periodof time (a course of treatment). For example, the dose may beadministered every 2 months, every 6 weeks, monthly, biweekly, weekly,or daily, as appropriate, over a period of time to encompass at least 2doses, 3 doses, 5 doses, 10 doses, or more.

In one embodiment, the agent is administered in combination with anothertherapy for lupus or nephritis, e.g., corticosteroids or NSAIDs.

In some cases, the subject can be identified by performing a diagnosticassay described herein, e.g., by evaluating a urine sample from thesubject, e.g., by evaluating Tweak levels or expression of a biomarkerwhose expression is increased by Tweak in mesangial cells. Exemplarybiomarkers include RANTES, KC, and/or IP-10. Tweak or the biomarker canbe detected by a variety of methods, including an immunoassay.

The term “treating” refers to administering a therapy in an amount,manner, and/or mode effective to improve or prevent a condition,symptom, or parameter associated with a disorder or to prevent onset,progression, or exacerbation of the disorder (including secondary damagecaused by the disorder), to either a statistically significant degree orto a degree detectable to one skilled in the art. Accordingly, treatingcan achieve therapeutic and/or prophylactic benefits. An effectiveamount, manner, or mode can vary depending on the subject and may betailored to the subject.

In another aspect, the invention features a method of evaluating acompound, e.g., screening for a compound, that modulates renal function.The method includes contacting a test compound to a Tweak or TweakRprotein, a cell, a tissue or a test subject, e.g., a non-human mammal,and evaluating the protein, cell, tissue or test subject for Tweak orTweakR expression or function. A test compound that decreases orinhibits Tweak or TweakR expression or function is identified as acompound that modulates renal function, e.g., a compound useful to treatnephritis. In one embodiment, the test compound interacts with Tweak orTweakR, directly or indirectly. In a preferred embodiment, the testcompound is a small molecule; a protein or peptide; an antibody; and/ora nucleotide sequence. For example, the agent can be an agent identifiedthrough a library screen.

In some embodiments, the method may include a two-step assay, e.g., afirst step of contacting and evaluating a test compound against a Tweakor TweakR protein or a Tweak expressing cell, and a second step ofcontacting and evaluating the test compound against a non-human animal.

The method can also include evaluating expression or activity (in thecell, tissue or non-human animal) of one or more biomarkers whoseexpression modulated (e.g., increased) by Tweak (e.g., RANTES, KC,and/or IP-10).

In another aspect, the disclosure features a method of making adiagnostic device. The method includes supplying a substrate, e.g., adipstick or other test device, well, tube, or strip; and adhering anreagent (e.g., an antibody) that detects one or more of Tweak, TweakR ora biomarker increased by Tweak, or providing such reagent as a solutionor other formulation available for use in the test device, e.g., in asandwich assay. In one embodiment, the reagent is applied by spraying,deposition of a liquid, or printing. The device can be supplied withinstruction for its use in the evaluation of kidney disease, e.g.,nephritis.

In another aspect, the invention features a computer readable recordencoded with (a) a subject identifier, e.g., a patient identifier, (b)one or more results from an evaluation of the subject, e.g., adiagnostic evaluation described herein, e.g., the level of expression,level or activity of Tweak or Tweak-R, in the subject, and optionally(c) a value for or related to renal disease (such as nephritis), e.g., avalue correlated with disease status or risk with regard to renaldisease. In one embodiment, the invention features a computer mediumhaving a plurality of digitally encoded data records. Each data recordincludes a value representing the expression, level or activity of Tweakor Tweak-R levels or activity, in a sample, and a descriptor of thesample. The descriptor of the sample can be an identifier of the sample,a subject from which the sample was derived (e.g., a patient), adiagnosis, or a treatment (e.g., a preferred treatment). In a preferredembodiment, the data record further includes values representing thelevel of expression, level or activity of genes other than Tweak orTweak-R (e.g., other genes associated with renal disease, or other geneson an array). The data record can be structured as a table, e.g., atable that is part of a database such as a relational database (e.g., aSQL database of the Oracle or Sybase database environments). Theinvention also includes a method of communicating information about asubject, e.g., by transmitting information, e.g., transmitting acomputer readable record described herein, e.g., over a computernetwork.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that uTWEAK levels correlate with lupus nephritis (LN)activity. A) comparison of uTWEAK levels between 43 patients with activerenal disease (defined as patients with rSLEDAI score of to 35 patientswith non-active renal disease (including patients with no previous renalinvolvement and those with previous kidney involvement, but in which therenal disease is inactive, all with rSLEDAI score of 0). B) comparisonof rSLEDAI scores of 78 SLE patients with their levels of uTWEAK yieldsa positive (ρ=0.405), significant (P<0.001) correlation.

FIG. 2 shows that uTWEAK levels are higher during active disease,particularly renal flares. A) In a comparison between patientsundergoing a flare and those with stable disease among all SLE patients(n=49), the flaring patients had significantly higher uTWEAK levels.Bars indicate median values. B) In patients with renal disease, bothactive and inactive (n=35), there was also a trend toward higher uTWEAKlevels in flaring patients as opposed to chronic stable patients. Barsindicate median values.

FIG. 3 shows that uTWEAK levels are significantly higher in renal thanin non-renal flares. A comparison among flaring SLE patients (n=31),undergoing a renal flare or a non-renal one: patients undergoing a renalflare had significantly higher uTWEAK levels than the patientsundergoing a non-renal flare. Bars indicate median values.

FIG. 4 shows that uTWEAK fluctuations reflect changes in renal diseaseactivity. A) In 4 of 6 patients, uTWEAK levels increased while thepatient is undergoing a renal flare. Each line represents one patient'schange in uTWEAK levels between the 2 timepoints in which the sampleswere obtained. B) Three uTWEAK measurements in one patient over thecourse of a year show fluctuations in uTWEAK levels that follow thecourse of the patient's renal disease activity, as defined by therSLEDAI and the presence of a disease flare. C) uTWEAK levels over 6months match the patient's disease activity (as determined by predefinedcriteria), despite a stable rSLEDAI score. For B-C: The solid line showsuTWEAK levels in pg/mg Cr, and the grey bars depict rSLEDAI scores.

FIG. 5 shows that uTWEAK levels correlate with other biomarkers. A)uTWEAK levels significantly correlated with uMCP-1 levels, ρ=0.501,P<0.001, n=51. uTWEAK levels in 80 SLE patients showed negativecorrelation with: B) standardized serum C3 levels, ρ=−0.262, P=0.019,and: C) Standardized serum C4 levels, ρ=−0.269, P=0.016. D) uTWEAKlevels correlated with standardized anti-dsDNA levels, ρ=0.459, P=0.008,n=32.

DETAILED DESCRIPTION

It has been found that increased Tweak or TweakR urinary, serum, plasmaor CSF levels correlate with increased severity, stage and/or activityof certain disorders. The methods described herein provide, inter alia,simple, quick, relatively non-invasive techniques for evaluating,staging, and/or diagnosing a subject for a disease, e.g., aninflammatory condition, e.g., before, during and/or after a treatment isbegun. The condition can be, e.g., nephritis (e.g., lupus nephritis),RA, PsA, lupus, fibrosis, cancer, or a neurodegenerative disease (e.g.,a neurodegenerative disease described herein).

Detection and Diagnosis of Nephritis

Although nephritis is discussed herein below as an exemplary disorderrelating to the methods described herein, it is understood that theetiology and clinical characteristics of other conditions describedherein are known to the skilled practitioner.

Nephritis is an inflammation of the kidneys. The two most common causesof nephritis are infection and auto-immune processes. Nephritis can be asymptom of underlying conditions such as systemic lupus erythematosus(SLE), diabetes, renal tuberculosis, or yellow fever. Lupus nephritis isan inflammation of the kidney caused by SLE. At least three potentiallyoverlapping, immuno-pathogenic mechanisms are supported by experimentaldata. First, circulating immune complexes consisting chiefly of DNA andanti-DNA are deposited in the kidney. Resulting complement activationand chemotaxis of neutrophils leads to a local inflammatory process.Second, in situ formation of antigen and antibody complexes maysimilarly lead to complement activation and leukocyte mediated injury.Third, antibodies against specific cellular targets may produce renalinjury. For example, antibodies, such as anti-ribosomal P, may bind tocytoplasmic antigens that have been translocated to the cell membranewith subsequent penetration and disruption of cellular function.

Glomerulonephritis is the most common type of nephritis and can includenephritic syndrome, nephrotic syndrome, and/or asymptomatic proteinuriaand hematuria syndrome, all of which may lead to end stage renal disease(ESRD) and kidney failure. Lupus nephritis can involve various internalstructures of the kidney and can include interstitial nephritis,glomerulonephritis (GN), mesangial GN, membranous GN, diffuseproliferative GN and/or membranoproliferative GN.

Nephritis can be detected or diagnosed by a variety of techniques,including urinalysis, e.g., detection of protein, casts, and/or redblood cells present in the urine; and/or BUN and/or creatinine tests toassess kidney function. Indications of lupus nephritis can also includehigh anti-DNA levels, high anti-dsDNA levels, low complement levels,high anti-nuclear antibody (ANA) panel titers and/or a positive lupuserythematosus test. Kidney X-rays or other imaging techniques and/or akidney biopsy may also be performed. Any of these tests may be used inaddition to the methods described herein.

Lupus nephritis (LN) remains a major cause of morbidity and mortality inSLE patients. Although the definition of renal involvement varies, overtrenal disease is found in at least one-third to one-half of SLEpatients, with reports of 5-year renal survival with treatment rangingfrom 46-95%. Early diagnosis and prompt treatment, however, maysignificantly improve long-term prognosis.

Lupus nephritis has been traditionally divided into six classes, definedby the World Health Organization (WHO) in 1982 (Churg and Sonbin.Classification and Atlas of Glomerular Disease. Tokyo: Igaku-Shoin;1982) which take into account the renal histopathological changestogether with activity of the nephritis. The prognosis and treatment ofLN is heavily dependent upon this disease classification. Currently, themost accurate and reliable method to diagnose and prognosticate LN, bothin terms of the activity and the chronicity of the renal processes, isby performing a biopsy. However, kidney biopsy, being an invasiveprocedure, can be accompanied by significant morbidity, and therefore isnot usually performed serially. Furthermore, with an essentially “blind”needle biopsy there can be a question of how representative the limitednumber of glomeruli usually obtained are of the status of the kidney.

Evaluating a Subject for Tweak or Fn14

Techniques for evaluating a subject for Tweak or Fn14 (or otherbiomarker described herein) in a biological sample of the subject areknown in the art. Such techniques can include detecting the presence,levels, expression or activity of a Tweak or Tweak-R protein, e.g., byqualitative or quantitative analysis of mRNA, cDNA, or protein, or byevaluating one or more nucleotides in a nucleic acid (genomic, mRNA, orcDNA) encoding Tweak or Tweak-R. Such techniques include methods forprotein detection (e.g., Western blot or ELISA), and hybridization-basedmethods for nucleic acid detection (e.g., PCR or Northern blot). Forexample, an immunoassay can be used to detect Tweak protein, e.g., in aurine sample of the subject. In other embodiments, the method caninclude administering a labeled Tweak or Tweak-R binding agent (e.g., anantibody) to a subject, and evaluating localization of the labeledbinding agent in the subject, e.g., by imaging the subject (e.g.,imaging at least a portion of the kidney of the subject).

Kits:

A kit can be used for assaying the Tweak pathway for risk, presence,stage or severity of a condition described herein, e.g., nephritisand/or lupus nephritis. The kit includes one or more reagents (e.g., ananti-Tweak antibody) capable of detecting one or more of: Tweak, TweakRor a biomarker described herein, in a biological sample of a subject,e.g., a human; and instructions for using the reagent to evaluate risk,predisposition, or prognosis for renal inflammation in a subject. Such akit can include instructions to use (e.g., to contact the agent) with asample from a subject (preferably a human) having lupus or otherinflammatory disorder, or risk therefor. In a preferred embodiment, theinstructions comprise instructions for use by or for a subject who hasnormal kidney function as defined by a clinical measure. Theinstructions can include directions to contact the agent with a urinesample of the subject.

The reagent can be attached to a solid substrate, e.g., a microtiterwell, a tube, a sheet (e.g., a nitrocellulose sheet), a dipstick orother test device. Preferably, the kit includes a dipstick or other testdevice. In a preferred embodiment, the reagent is an antibody or otherbinding protein. The antibody can be attached to a detectable label,e.g., an enzyme, a colorimetric reagent, a fluorescent substance, or aradioactive isotope. The kit can include a positive and/or a negativecontrol (e.g., a sample that includes an appropriate concentration ofTweak or the biomarker), e.g., with a stabilizer and/or preservative.The kit can also include a densitometer, or electrochemical strip.

Information about evaluating a subject can be obtained in a method thatincludes: supplying a test substrate (e.g., a tube, a strip, a dipstick,other test device, or a well) to which is attached an agent capable ofdetecting one or more of: Tweak or a biomarker described herein; andsupplying instructions to contact the test substrate with a subject'surine. The method optionally includes supplying instructions forreading, evaluating or interpreting the contacted substrate, e.g., toevaluate risk for, predisposition, or presence of renal inflammation.The method can be performed by health care provider or a third person,or by the subject.

Other possible approaches include the use of electrochemical sensorstrips, such as those used for home glucose testing, onto which a sampleis placed, and which strips include reagents for initiating a reactionwhen wetted by the sample. The sensor strip is inserted into a meterthat measures, e.g., diffusion-limited current of a reaction speciesindicative of the analyte of interest, e.g., Tweak and a biomarker whoseexpression modulated (e.g., increased) by Tweak (e.g., RANTES, KC,and/or IP-10). The meter then yields a display indicative of theconcentration of analyte in the sample.

Arrays:

Arrays are also particularly useful molecular tools for characterizing asample, e.g., a sample from a subject. For example, an array havingcapture probes for multiple genes, including probes for Tweak and/orother biomarkers, or for multiple proteins, can be used in a methoddescribed herein. Altered expression of Tweak nucleic acids and/orprotein can be used to evaluate a sample, e.g., a sample from a subject,e.g., to evaluate a disorder described herein.

Arrays can have many addresses, e.g., locatable sites, on a substrate.The featured arrays can be configured in a variety of formats,non-limiting examples of which are described below. The substrate can beopaque, translucent, or transparent. The addresses can be distributed,on the substrate in one dimension, e.g., a linear array; in twodimensions, e.g., a planar array; or in three dimensions, e.g., a threedimensional array. The solid substrate may be of any convenient shape orform, e.g., square, rectangular, ovoid, or circular.

Arrays can be fabricated by a variety of methods, e.g.,photolithographic methods (see, e.g., U.S. Pat. Nos. 5,143,854;5,510,270; and 5,527,681), mechanical methods (e.g., directed-flowmethods as described in U.S. Pat. No. 5,384,261), pin based methods(e.g., as described in U.S. Pat. No. 5,288,514), and bead basedtechniques (e.g., as described in PCT US/93/04145).

The capture probe can be a single-stranded nucleic acid, adouble-stranded nucleic acid (e.g., which is denatured prior to orduring hybridization), or a nucleic acid having a single-stranded regionand a double-stranded region. Preferably, the capture probe issingle-stranded. The capture probe can be selected by a variety ofcriteria, and preferably is designed by a computer program withoptimization parameters. The capture probe can be selected to hybridizeto a sequence rich (e.g., non-homopolymeric) region of the gene. The Tmof the capture probe can be optimized by prudent selection of thecomplementarity region and length. Ideally, the Tm of all capture probeson the array is similar, e.g., within 20, 10, 5, 3, or 2° C. of oneanother.

The isolated nucleic acid is preferably mRNA that can be isolated byroutine methods, e.g., including DNase treatment to remove genomic DNAand hybridization to an oligo-dT coupled solid substrate (e.g., asdescribed in Current Protocols in Molecular Biology, John Wiley & Sons,N.Y). The substrate is washed, and the mRNA is eluted.

The isolated mRNA can be reversed transcribed and optionally amplified,e.g., by rtPCR, e.g., as described in (U.S. Pat. No. 4,683,202). Thenucleic acid can be an amplification product, e.g., from PCR (U.S. Pat.Nos. 4,683,196 and 4,683,202); rolling circle amplification (“RCA,” U.S.Pat. No. 5,714,320), isothermal RNA amplification or NASBA (U.S. Pat.Nos. 5,130,238; 5,409,818; and 5,554,517), and strand displacementamplification (U.S. Pat. No. 5,455,166). The nucleic acid can be labeledduring amplification, e.g., by the incorporation of a labelednucleotide. Examples of preferred labels include fluorescent labels,e.g., red-fluorescent dye Cy5 (Amersham) or green-fluorescent dye Cy3(Amersham), and chemiluminescent labels, e.g., as described in U.S. Pat.No. 4,277,437. Alternatively, the nucleic acid can be labeled withbiotin, and detected after hybridization with labeled streptavidin,e.g., streptavidin-phycoerythrin (Molecular Probes).

The labeled nucleic acid can be contacted to the array. In addition, acontrol nucleic acid or a reference nucleic acid can be contacted to thesame array. The control nucleic acid or reference nucleic acid can belabeled with a label other than the sample nucleic acid, e.g., one witha different emission maximum. Labeled nucleic acids can be contacted toan array under hybridization conditions. The array can be washed, andthen imaged to detect fluorescence at each address of the array.

The expression level of a Tweak or other biomarker can be determinedusing an antibody specific for the polypeptide (e.g., using a westernblot or an ELISA assay). Moreover, the expression levels of multipleproteins, including Tweak and the exemplary biomarkers provided herein,can be rapidly determined in parallel using a polypeptide array havingantibody capture probes for each of the polypeptides. Antibodiesspecific for a polypeptide can be generated by a method described herein(see “Antibody Generation”). The expression level of a TWEAK and theexemplary biomarkers provided herein can be measured in a subject (e.g.,in vivo imaging) or in a biological sample from a subject (e.g., blood,serum, plasma, or synovial fluid).

A low-density (96 well format) protein array has been developed in whichproteins are spotted onto a nitrocellulose membrane (Ge (2000) NucleicAcids Res. 28, e3, I-VII). A high-density protein array (100,000 sampleswithin 222×222 mm) used for antibody screening was formed by spottingproteins onto polyvinylidene difluoride (PVDF) (Lueking et al. (1999)Anal. Biochem. 270:103-111). See also, e.g., Mendoza et al. (1999).Biotechniques 27:778-788; MacBeath and Schreiber (2000) Science289:1760-1763; and De Wildt et al. (2000). Nature Biotech. 18:989-994.These art-known methods and other can be used to generate an array ofantibodies for detecting the abundance of polypeptides in a sample. Thesample can be labeled, e.g., biotinylated, for subsequent detection withstreptavidin coupled to a fluorescent label. The array can then bescanned to measure binding at each address.

The nucleic acid and polypeptide arrays of the invention can be used inwide variety of applications. For example, the arrays can be used toanalyze a patient sample. The sample is compared to data obtainedpreviously, e.g., known clinical specimens or other patient samples.Further, the arrays can be used to characterize a cell culture sample,e.g., to determine a cellular state after varying a parameter, e.g.,exposing the cell culture to an antigen, a transgene, or a testcompound.

The expression data can be stored in a database, e.g., a relationaldatabase such as a SQL database (e.g., Oracle or Sybase databaseenvironments). The database can have multiple tables. For example, rawexpression data can be stored in one table, wherein each columncorresponds to a gene being assayed, e.g., an address or an array, andeach row corresponds to a sample. A separate table can store identifiersand sample information, e.g., the batch number of the array used, date,and other quality control information.

Expression profiles obtained from gene expression analysis on an arraycan be used to compare samples and/or cells in a variety of states asdescribed in Golub et al. ((1999) Science 286:531). In one embodiment,expression (e.g., mRNA expression or protein expression) information fora gene encoding TWEAK and/or a gene encoding a exemplary biomarkerprovided herein are evaluated, e.g., by comparison a reference value,e.g., a reference value. Reference values can be obtained from acontrol, a reference subject. Reference values can also be obtained fromstatistical analysis, e.g., to provide a reference value for a cohort ofsubject, e.g., age and gender matched subject, e.g., normal subjects orsubject who have rheumatoid arthritis or other disorder describedherein. Statistical similarity to a particular reference (e.g., to areference for a risk-associated cohort) or a normal cohort can be usedto provide an assessment (e.g., an indication of risk of inflammatorydisorder) to a subject, e.g., a subject who has not been diagnosed witha disorder described herein.

Subjects suitable for treatment can also be evaluated for expressionand/or activity of TWEAK and/or other biomarker. Subjects can beidentified as suitable for treatment (e.g., with a TWEAK blockingagent), if the expression and/or activity for TWEAK and/or the otherbiomarker is elevated relative to a reference, e.g., reference value,e.g., a reference value associated with normal.

Subjects who are being administered an agent described herein or othertreatment can be evaluated as described for expression and/or activityof TWEAK and/or other biomarkers described herein. The subject can beevaluated at multiple times. e.g., at multiple times during a course oftherapy, e.g., during a therapeutic regimen. Treatment of the subjectcan be modified depending on how the subject is responding to thetherapy. For example, a reduction in TWEAK expression or activity or areduction in the expression or activity of genes stimulated by TWEAK canbe indicative of responsiveness.

Particular effects mediated by an agent may show a difference (e.g.,relative to an untreated subject, control subject, or other reference)that is statistically significant (e.g., P value<0.05 or 0.02).Statistical significance can be determined by any art known method.Exemplary statistical tests include: the Students T-test, Mann Whitney Unon-parametric test, and Wilcoxon non-parametric statistical test. Somestatistically significant relationships have a P value of less than 0.05or 0.02.

Any combination of the above methods can also be used. The above methodscan be used to evaluate any genetic locus, e.g., in a method foranalyzing genetic information from particular groups of individuals orin a method for analyzing a polymorphism associated with a disorderdescribed herein, e.g., rheumatoid arthritis, e.g., in a gene encodingTWEAK or another biomarker described herein.

Tweak-Tweak Receptor Blocking Agents

Tweak pathway inhibitors to treat nephritis include Tweak/Tweak-Rblocking agents. The agent may be any type of compound (e.g., smallorganic or inorganic molecule, nucleic acid, protein, or peptidemimetic) that can be administered to a subject.

In one embodiment, the blocking agent is a biologic, e.g., a proteinhaving a molecular weight of between 5-300 kDa. For example, aTweak/Tweak-R blocking agent may inhibit binding of Tweak to a Tweakreceptor. A typical Tweak/Tweak-R blocking agent can bind to Tweak or aTweak receptor, e.g., Fn14. A Tweak/Tweak-R blocking agent that binds toTweak may block the binding site on Tweak or a Tweak receptor, alter theconformation of Tweak or a Tweak receptor, or otherwise decrease theaffinity of Tweak for a Tweak receptor or prevent the interactionbetween Tweak and a Tweak receptor. A Tweak/Tweak-R blocking agent(e.g., an antibody) may bind to Tweak or to a Tweak receptor with a Kdof less than 10-6, 10-7, 10-8, 10-9, or 10-10 M. In one embodiment, theblocking agent binds to Tweak with an affinity at least 5, 10, 20, 50,100, 200, 500, or 1000 better than its affinity for TNF or another TNFsuperfamily member (other than Tweak). In one embodiment, the blockingagent binds to the Tweak receptor with an affinity at least 5, 10, 20,50, 100, 200, 500, or 1000-fold better than its affinity for the TNFreceptor or a receptor for another TNF superfamily member. A preferredTweak/Tweak-R blocking agent specifically binds Tweak or Tweak-R, suchas a Tweak or Tweak-R specific antibody, e.g., a monoclonal antibody.

The sequence of human Tweak (SEQ ID NO:1) is shown below.

  1 MAARRSQRRR GRRGEPGTAL LVPLALGLGL ALACLGLLLA VVSLGSRASL SAQEPAQEEL 61 VAEEDQDPSE LNPQTEESQD PAPFLNRLVR PRRSAPKGRK TRARRAIAAH YEVHPRPGQD121 GAQAGVDGTV SGWEEARINS SSPLRYNRQI GEFIVTRAGL YYLYCQVHFD EGKAVYLKLD181 LLVDGVLALR CLEEFSATAA SSLGPQLRLC QVSGLLALRP GSSLRIRTLP WAHLKAAPFL241 TYFGLFQVH

Also included are proteins that include an amino acid sequence at least90, 92, 95, 97, 98, 99% identical and completely identical to the matureprocessed region of SEQ ID NO:1 (e.g., an amino acid sequence at least90, 92, 95, 97, 98, 99% identical or completely identical to amino acidsX1-249 of SEQ ID NO:1, where amino acid X1 is selected from the group ofresidues 75-115 of SEQ ID NO:1, e.g., X1 is residue Arg 93 of SEQ IDNO:1) and proteins encoded by a nucleic acid that hybridizes under highstringency conditions to the DNA encoding SEQ ID NO:1. Preferably, aTweak protein, in its processed mature form, is capable of providing atleast one Tweak activity, e.g., ability to activate Fn14 and/or celldeath in cortical neurons.

The sequence of human Tweak-R (SEQ ID NO:2) is shown below.

  1 MARGSLRRLL RLLVLGLWLA LLRSVAGEQA PGTAPCSRGS SWSADLDKCM DCASCRARPH 61 SDFCLGCAAA PPAPFRLLWP ILGGALSLTF VLGLLSGFLV WRRCRRREKF TTPIEETGGE121 GCPAVALIQ

Soluble proteins that include an amino acid sequence at least 90, 92,95, 97, 98, 99% identical to the extracellular domain of Fn14 (andTweak-binding fragments thereof) and proteins encoded by a nucleic acidthat hybridizes under high stringency conditions to a human Fn14 proteinare useful in the methods described herein. Preferably, a Fn14 proteinuseful in the methods described herein is a soluble Fn14 (lacking atransmembrane domain) that includes a region that binds to a Tweakprotein, e.g., an amino acid sequence at least 90, 92, 95, 97, 98, or99% identical, or completely identical, to amino acids 28-X1 of SEQ IDNO:2, where amino acid X1 is selected from the group of residues 68 to80 of SEQ ID NO:2.

Calculations of “homology” or “sequence identity” between two sequences(the terms are used interchangeably herein) are performed as follows.The sequences are aligned for optimal comparison purposes (e.g., gapscan be introduced in one or both of a first and a second amino acid ornucleic acid sequence for optimal alignment and non-homologous sequencescan be disregarded for comparison purposes). The optimal alignment isdetermined as the best score using the GAP program in the GCG softwarepackage with a Blossum 62 scoring matrix with a gap penalty of 12, a gapextend penalty of 4, and a frameshift gap penalty of 5. The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences.

As used herein, the term “hybridizes under high stringency conditions”describes conditions for hybridization and washing. Guidance forperforming hybridization reactions can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which isincorporated by reference. Aqueous and nonaqueous methods are describedin that reference and either can be used. High stringency hybridizationconditions include hybridization in 6×SSC at about 45° C., followed byone or more washes in 0.2×SSC, 0.1% SDS at 65° C., or substantiallysimilar conditions.

Exemplary Tweak/Tweak-R blocking agents include antibodies that bind toTweak or Tweak-R and soluble forms of the Tweak-R that compete with cellsurface Tweak-R for binding to Tweak. An example of a soluble form ofthe Tweak-R is an Fc fusion protein that includes at least a portion ofthe extracellular domain of Tweak-R (e.g., a soluble Tweak-bindingfragment of Tweak-R), referred to as Tweak-R-Fc. Other soluble forms ofTweak-R, e.g., forms that do not include an Fc domain, can also be used.Antibody blocking agents are further discussed below. Other types ofblocking agents, e.g., small molecules, nucleic acid or nucleicacid-based aptamers, and peptides, can be isolated by screening, e.g.,as described in Jhaveri et al. Nat. Biotechnol. 18:1293 and U.S. Pat.No. 5,223,409. Exemplary assays for determining if an agent binds toTweak or Tweak-R and for determining if an agent modulates aTweak/Tweak-R interaction are described, e.g., in US 2004-0033225.

An exemplary soluble form of the Tweak-R protein includes a region ofthe Tweak-R protein that binds to Tweak, e.g., about amino acids 32-75,31-75, 31-78, or 28-79 of SEQ ID NO:2. This region can be physicallyassociated, e.g., fused to another amino acid sequence, e.g., an Fcdomain, at its N- or C-terminus. The region from Tweak-R can be spacedby a linker from the heterologous amino acid sequence. U.S. Pat. No.6,824,773 describes an exemplary Tweak-R fusion protein.

Antibodies

Exemplary Tweak/Tweak-R blocking agents include antibodies that bind toTweak and/or Tweak-R. In on embodiment, the antibody inhibits theinteraction between Tweak and a Tweak receptor, e.g., by physicallyblocking the interaction, decreasing the affinity of Tweak and/orTweak-R for its counterpart, disrupting or destabilizing Tweakcomplexes, sequestering Tweak or a Tweak-R, or targeting Tweak orTweak-R for degradation. In one embodiment, the antibody can bind toTweak or Tweak-R at one or more amino acid residues that participate inthe Tweak/Tweak-R binding interface. Such amino acid residues can beidentified, e.g., by alanine scanning. In another embodiment, theantibody can bind to residues that do not participate in theTweak/Tweak-R binding. For example, the antibody can alter aconformation of Tweak or Tweak-R and thereby reduce binding affinity, orthe antibody may sterically hinder Tweak/Tweak-R binding. In oneembodiment, the antibody can prevent activation of a Tweak/Tweak-Rmediated event or activity (e.g., NF-kappaB activation).

As used herein, the term “antibody” refers to a protein that includes atleast one immunoglobulin variable region, e.g., an amino acid sequencethat provides an immunoglobulin variable domain or an immunoglobulinvariable domain sequence. For example, an antibody can include a heavy(H) chain variable region (abbreviated herein as VH), and a light (L)chain variable region (abbreviated herein as VL). In another example, anantibody includes two heavy (H) chain variable regions and two light (L)chain variable regions. The term “antibody” encompasses antigen-bindingfragments of antibodies (e.g., single chain antibodies, Fab fragments,F(ab′)2 fragments, Fd fragments, Fv fragments, and dAb fragments) aswell as complete antibodies, e.g., intact and/or full lengthimmunoglobulins of types IgA, IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgE,IgD, IgM (as well as subtypes thereof). The light chains of theimmunoglobulin may be of types kappa or lambda. In one embodiment, theantibody is glycosylated. An antibody can be functional forantibody-dependent cytotoxicity and/or complement-mediated cytotoxicity,or may be non-functional for one or both of these activities.

The VH and VL regions can be further subdivided into regions ofhypervariability, termed “complementarity determining regions” (“CDR”),interspersed with regions that are more conserved, termed “frameworkregions” (FR). The extent of the FR's and CDR's has been preciselydefined (see, Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, US Department of Health and HumanServices, NIH Publication No. 91-3242; and Chothia, C. et al. (1987) J.Mol. Biol. 196:901-917). Kabat definitions are used herein. Each VH andVL is typically composed of three CDR's and four FR's, arranged fromamino-terminus to carboxyl-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4.

An “immunoglobulin domain” refers to a domain from the variable orconstant domain of immunoglobulin molecules. Immunoglobulin domainstypically contain two beta-sheets formed of about seven beta-strands,and a conserved disulphide bond (see, e.g., A. F. Williams and A. N.Barclay (1988) Ann. Rev Immunol. 6:381-405). An “immunoglobulin variabledomain sequence” refers to an amino acid sequence that can form astructure sufficient to position CDR sequences in a conformationsuitable for antigen binding. For example, the sequence may include allor part of the amino acid sequence of a naturally occurring variabledomain. For example, the sequence may omit one, two or more N- orC-terminal amino acids, internal amino acids, may include one or moreinsertions or additional terminal amino acids, or may include otheralterations. In one embodiment, a polypeptide that includes animmunoglobulin variable domain sequence can associate with anotherimmunoglobulin variable domain sequence to form a target bindingstructure (or “antigen binding site”), e.g., a structure that interactswith Tweak or a Tweak receptor.

The VH or VL chain of the antibody can further include all or part of aheavy or light chain constant region, to thereby form a heavy or lightimmunoglobulin chain, respectively. In one embodiment, the antibody is atetramer of two heavy immunoglobulin chains and two light immunoglobulinchains. The heavy and light immunoglobulin chains can be connected bydisulfide bonds. The heavy chain constant region typically includesthree constant domains, CH1, CH2, and CH3. The light chain constantregion typically includes a CL domain. The variable region of the heavyand light chains contains a binding domain that interacts with anantigen. The constant regions of the antibodies typically mediate thebinding of the antibody to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (Clq) of the classical complement system.

One or more regions of an antibody can be human, effectively human, orhumanized. For example, one or more of the variable regions can be humanor effectively human. For example, one or more of the CDRs, e.g., HCCDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3, can be human.Each of the light chain CDRs can be human. HC CDR3 can be human. One ormore of the framework regions can be human, e.g., FR1, FR2, FR3, and FR4of the HC or LC. In one embodiment, all the framework regions are human,e.g., derived from a human somatic cell, e.g., a hematopoietic cell thatproduces immunoglobulins or a non-hematopoietic cell. In one embodiment,the human sequences are germline sequences, e.g., encoded by a germlinenucleic acid. One or more of the constant regions can be human,effectively human, or humanized. In another embodiment, at least 70, 75,80, 85, 90, 92, 95, or 98% of the framework regions (e.g., FR1, FR2, andFR3, collectively, or FR1, FR2, FR3, and FR4, collectively) or theentire antibody can be human, effectively human, or humanized. Forexample, FR1, FR2, and FR3 collectively can be at least 70, 75, 80, 85,90, 92, 95, 98, or 99% identical, or completely identical, to a humansequence encoded by a human germline segment.

An “effectively human” immunoglobulin variable region is animmunoglobulin variable region that includes a sufficient number ofhuman framework amino acid positions such that the immunoglobulinvariable region does not elicit an immunogenic response in a normalhuman. An “effectively human” antibody is an antibody that includes asufficient number of human amino acid positions such that the antibodydoes not elicit an immunogenic response in a normal human.

A “humanized” immunoglobulin variable region is an immunoglobulinvariable region that is modified such that the modified form elicitsless of an immune response in a human than does the non-modified form,e.g., is modified to include a sufficient number of human frameworkamino acid positions such that the immunoglobulin variable region doesnot elicit an immunogenic response in a normal human. Descriptions of“humanized” immunoglobulins include, for example, U.S. Pat. Nos.6,407,213 and 5,693,762. In some cases, humanized immunoglobulins caninclude a non-human amino acid at one or more framework amino acidpositions.

Antibody Generation

Antibodies that bind to Tweak or a Tweak-R can be generated by a varietyof means, including immunization, e.g., using an animal, or in vitromethods such as phage display. All or part of Tweak or a Tweak receptorcan be used as an immunogen or as a target for selection. For example,Tweak or a fragment thereof, Tweak-R or a fragment thereof, can be usedas an immunogen. In one embodiment, the immunized animal containsimmunoglobulin producing cells with natural, human, or partially humanimmunoglobulin loci. In one embodiment, the non-human animal includes atleast a part of a human immunoglobulin gene. For example, it is possibleto engineer mouse strains deficient in mouse antibody production withlarge fragments of the human Ig loci. Using the hybridoma technology,antigen-specific monoclonal antibodies derived from the genes with thedesired specificity may be produced and selected. See, e.g., XENOMOUSE™,Green et al. (1994) Nat. Gen. 7:13-21; US 2003-0070185; U.S. Pat. No.5,789,650; and WO 96/34096.

Non-human antibodies to Tweak or a Tweak receptor can also be produced,e.g., in a rodent. The non-human antibody can be humanized, e.g., asdescribed in EP 239 400; U.S. Pat. Nos. 6,602,503; 5,693,761; and6,407,213, deimmunized, or otherwise modified to make it effectivelyhuman.

EP 239 400 (Winter et al.) describes altering antibodies by substitution(within a given variable region) of their complementarity determiningregions (CDRs) for one species with those from another. Typically, CDRsof a non-human (e.g., murine) antibody are substituted into thecorresponding regions in a human antibody by using recombinant nucleicacid technology to produce sequences encoding the desired substitutedantibody. Human constant region gene segments of the desired isotype(usually gamma I for CH and kappa for CL) can be added and the humanizedheavy and light chain genes can be co-expressed in mammalian cells toproduce soluble humanized antibody. Other methods for humanizingantibodies can also be used. For example, other methods can account forthe three dimensional structure of the antibody, framework positionsthat are in three dimensional proximity to binding determinants, andimmunogenic peptide sequences. See, e.g., WO 90/07861; U.S. Pat. Nos.5,693,762; 5,693,761; 5,585,089; and 5,530,101; Tempest et al. (1991)Biotechnology 9:266-271 and U.S. Pat. No. 6,407,213. Still anothermethod is termed “humaneering” and is described, for example, in US2005-008625.

Fully human monoclonal antibodies that bind to Tweak or a Tweak receptorcan be produced, e.g., using in vitro-primed human splenocytes, asdescribed by Boerner et al. (1991) J. Immunol. 147:86-95. They may beprepared by repertoire cloning as described by Persson et al. (1991)Proc. Nat. Acad. Sci. USA 88:2432-2436 or by Huang and Stollar (1991) J.Immunol. Methods 141:227-236; also U.S. Pat. No. 5,798,230. Largenonimmunized human phage display libraries may also be used to isolatehigh affinity antibodies that can be developed as human therapeuticsusing standard phage technology (see, e.g., Hoogenboom et al. (1998)Immunotechnology 4:1-20; Hoogenboom et al. (2000) Immunol Today 2:371-8;and US 2003-0232333).

Antibody and Protein Production

Antibodies and other proteins described herein can be produced inprokaryotic and eukaryotic cells. In one embodiment, the antibodies(e.g., scFv's) are expressed in a yeast cell such as Pichia (see, e.g.,Powers et al. (2001) J. Immunol. Methods 251:123-35), Hanseula, orSaccharomyces.

Antibodies, particularly full length antibodies, e.g., IgGs, can beproduced in mammalian cells. Exemplary mammalian host cells forrecombinant expression include Chinese Hamster Ovary (CHO cells)(including dhfr-CHO cells, described in Urlaub and Chasin (1980) Proc.Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker,e.g., as described in Kaufman and Sharp (1982) Mol. Biol. 159:601-621),lymphocytic cell lines, e.g., NS0 myeloma cells and SP2 cells, COScells, K562, and a cell from a transgenic animal, e.g., a transgenicmammal. For example, the cell is a mammary epithelial cell.

In addition to the nucleic acid sequence encoding the immunoglobulindomain, the recombinant expression vectors may carry additional nucleicacid sequences, such as sequences that regulate replication of thevector in host cells (e.g., origins of replication) and selectablemarker genes. The selectable marker gene facilitates selection of hostcells into which the vector has been introduced (see e.g., U.S. Pat.Nos. 4,399,216; 4,634,665; and 5,179,017). Exemplary selectable markergenes include the dihydrofolate reductase (DHFR) gene (for use indhfr-host cells with methotrexate selection/amplification) and the neogene (for G418 selection).

In an exemplary system for recombinant expression of an antibody (e.g.,a full length antibody or an antigen-binding portion thereof), arecombinant expression vector encoding both the antibody heavy chain andthe antibody light chain is introduced into dhfr-CHO cells by calciumphosphate-mediated transfection. Within the recombinant expressionvector, the antibody heavy and light chain genes are each operativelylinked to enhancer/promoter regulatory elements (e.g., derived fromSV40, CMV, adenovirus and the like, such as a CMV enhancer/AdMLPpromoter regulatory element or an SV40 enhancer/AdMLP promoterregulatory element) to drive high levels of transcription of the genes.The recombinant expression vector also carries a DHFR gene, which allowsfor selection of CHO cells that have been transfected with the vectorusing methotrexate selection/amplification. The selected transformanthost cells are cultured to allow for expression of the antibody heavyand light chains and intact antibody is recovered from the culturemedium. Standard molecular biology techniques are used to prepare therecombinant expression vector, to transfect the host cells, to selectfor transformants, to culture the host cells, and to recover theantibody from the culture medium. For example, some antibodies can beisolated by affinity chromatography with a Protein A or Protein G.

Antibodies (and Fc fusions) may also include modifications, e.g.,modifications that alter Fc function, e.g., to decrease or removeinteraction with an Fc receptor or with Clq, or both. For example, thehuman IgG1 constant region can be mutated at one or more residues, e.g.,one or more of residues 234 and 237, e.g., according to the numbering inU.S. Pat. No. 5,648,260. Other exemplary modifications include thosedescribed in U.S. Pat. No. 5,648,260.

For some proteins that include an Fc domain, the antibody/proteinproduction system may be designed to synthesize antibodies or otherproteins in which the Fc region is glycosylated. For example, the Fcdomain of IgG molecules is glycosylated at asparagine 297 in the CH2domain. The Fc domain can also include other eukaryoticpost-translational modifications. In other cases, the protein isproduced in a form that is not glycosylated.

Antibodies and other proteins can also be produced by a transgenicanimal. For example, U.S. Pat. No. 5,849,992 describes a method forexpressing an antibody in the mammary gland of a transgenic mammal. Atransgene is constructed that includes a milk-specific promoter andnucleic acid sequences encoding the antibody of interest, e.g., anantibody described herein, and a signal sequence for secretion. The milkproduced by females of such transgenic mammals includes,secreted-therein, the protein of interest, e.g., an antibody or Fcfusion protein. The protein can be purified from the milk, or for someapplications, used directly.

Methods described in the context of antibodies can be adapted to otherproteins, e.g., Fc fusions and soluble receptor fragments.

Nucleic Acid Antagonists

In certain implementations, nucleic acid antagonists are used todecrease expression of an endogenous gene encoding Tweak or a Tweak-R,e.g., Fn14. In one embodiment, the nucleic acid antagonist is an siRNAthat targets mRNA encoding Tweak or a Tweak-R. Other types ofantagonistic nucleic acids can also be used, e.g., an siRNA, a ribozyme,a triple-helix former, an aptamer, or an antisense nucleic acid.

siRNAs are small double stranded RNAs (dsRNAs) that optionally includeoverhangs. For example, the duplex region of an siRNA is about 18 to 25nucleotides in length, e.g., about 19, 20, 21, 22, 23, or 24 nucleotidesin length. Typically, the siRNA sequences are exactly complementary tothe target mRNA. dsRNAs and siRNAs in particular can be used to silencegene expression in mammalian cells (e.g., human cells). See, e.g.,Clemens et al. (2000) Proc. Natl. Acad. Sci. USA 97:6499-6503; Billy etal. (2001) Proc. Natl. Sci. USA 98:14428-14433; Elbashir et al. (2001)Nature. 411:494-8; Yang et al. (2002) Proc. Natl. Acad. Sci. USA99:9942-9947, U.S. 20030166282, 20030143204, 20040038278, and20030224432.

Anti-sense agents can include, for example, from about 8 to about 80nucleobases (i.e. from about 8 to about 80 nucleotides), e.g., about 8to about 50 nucleobases, or about 12 to about 30 nucleobases. Anti-sensecompounds include ribozymes, external guide sequence (EGS)oligonucleotides (oligozymes), and other short catalytic RNAs orcatalytic oligonucleotides which hybridize to the target nucleic acidand modulate its expression. Anti-sense compounds can include a stretchof at least eight consecutive nucleobases that are complementary to asequence in the target gene. An oligonucleotide need not be 100%complementary to its target nucleic acid sequence to be specificallyhybridizable. An oligonucleotide is specifically hybridizable whenbinding of the oligonucleotide to the target interferes with the normalfunction of the target molecule to cause a loss of utility, and there isa sufficient degree of complementarity to avoid non-specific binding ofthe oligonucleotide to non-target sequences under conditions in whichspecific binding is desired, i.e., under physiological conditions in thecase of in vivo assays or therapeutic treatment or, in the case of invitro assays, under conditions in which the assays are conducted.

Hybridization of antisense oligonucleotides with mRNA (e.g., an mRNAencoding Tweak or Tweak-R) can interfere with one or more of the normalfunctions of mRNA. The functions of mRNA to be interfered with includeall key functions such as, for example, translocation of the RNA to thesite of protein translation, translation of protein from the RNA,splicing of the RNA to yield one or more mRNA species, and catalyticactivity which may be engaged in by the RNA. Binding of specificprotein(s) to the RNA may also be interfered with by antisenseoligonucleotide hybridization to the RNA.

Exemplary antisense compounds include DNA or RNA sequences thatspecifically hybridize to the target nucleic acid, e.g., the mRNAencoding Tweak or Tweak-R. The complementary region can extend forbetween about 8 to about 80 nucleobases. The compounds can include oneor more modified nucleobases. Modified nucleobases may include, e.g.,5-substituted pyrimidines such as 5-iodouracil, 5-iodocytosine, andC5-propynyl pyrimidines such as C5-propynylcytosine andC5-propynyluracil. Other suitable modified nucleobases includeN4-(C1-C12) alkylaminocytosines and N4,N4-(C1-C12)dialkylaminocytosines. Modified nucleobases may also include7-substituted-8-aza-7-deazapurines and 7-substituted-7-deazapurines suchas, for example, 7-iodo-7-deazapurines, 7-cyano-7-deazapurines,7-aminocarbonyl-7-deazapurines. Examples of these include6-amino-7-iodo-7-deazapurines, 6-amino-7-cyano-7-deazapurines,6-amino-7-aminocarbonyl-7-deazapurines,2-amino-6-hydroxy-7-iodo-7-deazapurines,2-amino-6-hydroxy-7-cyano-7-deazapurines, and2-amino-6-hydroxy-7-aminocarbonyl-7-deazapurines. Furthermore,N6-(C1-C12) alkylaminopurines and N6,N6-(C1-C12) dialkylaminopurines,including N6-methylaminoadenine and N6,N6-dimethylaminoadenine, are alsosuitable modified nucleobases. Similarly, other 6-substituted purinesincluding, for example, 6-thioguanine may constitute appropriatemodified nucleobases. Other suitable nucleobases include 2-thiouracil,8-bromoadenine, 8-bromoguanine, 2-fluoroadenine, and 2-fluoroguanine.Derivatives of any of the aforementioned modified nucleobases are alsoappropriate. Substituents of any of the preceding compounds may includeC1-C30 alkyl, C2-C30 alkenyl, C2-C30 alkynyl, aryl, aralkyl, heteroaryl,halo, amino, amido, nitro, thio, sulfonyl, carboxyl, alkoxy,alkylcarbonyl, alkoxycarbonyl, and the like.

Descriptions of other types of nucleic acid agents are also available.See, e.g., U.S. Pat. Nos. 4,987,071; 5,116,742; and 5,093,246; Woolf etal. (1992) Proc Natl Acad Sci USA; Antisense RNA and DNA, D. A. Melton,Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1988);89:7305-9; Haselhoff and Gerlach (1988) Nature 334:585-59; Helene, C.(1991) Anticancer Drug Des. 6:569-84; Helene (1992) Ann. N.Y. Acad. Sci.660:27-36; and Maher (1992) Bioassays 14:807-15.

Artificial Transcription Factors

Artificial transcription factors can also be used to regulate expressionof Tweak and/or Tweak-R. The artificial transcription factor can bedesigned or selected from a library, e.g., for ability to bind to asequence in an endogenous gene encoding Tweak or Tweak-R, e.g., in aregulatory region, e.g., the promoter. For example, the artificialtranscription factor can be prepared by selection in vitro (e.g., usingphage display, U.S. Pat. No. 6,534,261) or in vivo, or by design basedon a recognition code (see, e.g., WO 00/42219 and U.S. Pat. No.6,511,808). See, e.g., Rebar et al. (1996) Methods Enzymol 267:129;Greisman and Pabo (1997) Science 275:657; Isalan et al. (2001) Nat.Biotechnol 19:656; and Wu et al. (1995) Proc. Natl. Acad. Sci. USA92:344 for, among other things, methods for creating libraries of variedzinc finger domains.

Optionally, an artificial transcription factor can be fused to atranscriptional regulatory domain, e.g., an activation domain toactivate transcription or a repression domain to repress transcription.In particular, repression domains can be used to decrease expression ofendogenous genes encoding Tweak or Tweak-R. The artificial transcriptionfactor can itself be encoded by a heterologous nucleic acid that isdelivered to a cell or the protein itself can be delivered to a cell(see, e.g., U.S. Pat. No. 6,534,261). The heterologous nucleic acid thatincludes a sequence encoding the artificial transcription factor can beoperably linked to an inducible promoter, e.g., to enable fine controlof the level of the artificial transcription factor in the cell.

Pharmaceutical Compositions

A Tweak/Tweak-R blocking agent (e.g., an antibody or soluble Tweak-Rprotein, e.g., Tweak-R-Fc) can be formulated as a pharmaceuticalcomposition, e.g., for administration to a subject to treat thenephritis. Typically, a pharmaceutical composition includes apharmaceutically acceptable carrier. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like that are physiologically compatible. Thecomposition can include a pharmaceutically acceptable salt, e.g., anacid addition salt or a base addition salt (see e.g., Berge, S. M., etal. (1977) J. Pharm. Sci. 66:1-19).

The Tweak/Tweak-R blocking agent can be formulated according to standardmethods. Pharmaceutical formulation is a well-established art, and isfurther described, e.g., in Gennaro (ed.), Remington: The Science andPractice of Pharmacy, 20th ed., Lippincott, Williams & Wilkins (2000)(ISBN: 0683306472); Ansel et al., Pharmaceutical Dosage Forms and DrugDelivery Systems, 7th Ed., Lippincott Williams & Wilkins Publishers(1999) (ISBN: 0683305727); and Kibbe (ed.), Handbook of PharmaceuticalExcipients American Pharmaceutical Association, 3rd ed. (2000) (ISBN:091733096X).

In one embodiment, the Tweak/Tweak-R blocking agent (e.g., an antibodyor Tweak-R-Fc) can be formulated with excipient materials, such assodium chloride, sodium dibasic phosphate heptahydrate, sodium monobasicphosphate, and a stabilizer. It can be provided, for example, in abuffered solution at a suitable concentration and can be stored at 2-8°C.

The pharmaceutical compositions may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, tablets, pills, powders, liposomes and suppositories.The preferred form can depend on the intended mode of administration andtherapeutic application. Typically compositions for the agents describedherein are in the form of injectable or infusible solutions.

Such compositions can be administered by a parenteral mode (e.g.,intravenous, subcutaneous, intraperitoneal, or intramuscular injection).The phrases “parenteral administration” and “administered parenterally”as used herein mean modes of administration other than enteral andtopical administration, usually by injection, and include, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

The composition can be formulated as a solution, microemulsion,dispersion, liposome, or other ordered structure suitable for stablestorage at high concentration. Sterile injectable solutions can beprepared by incorporating an agent described herein in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating anagent described herein into a sterile vehicle that contains a basicdispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying that yields a powder of an agentdescribed herein plus any additional desired ingredient from apreviously sterile-filtered solution thereof. The proper fluidity of asolution can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prolonged absorption ofinjectable compositions can be brought about by including in thecomposition an agent that delays absorption, for example, monostearatesalts and gelatin.

In certain embodiments, the Tweak/Tweak-R blocking agent may be preparedwith a carrier that will protect the compound against rapid release,such as a controlled release formulation, including implants, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known. See, e.g., Sustained and Controlled Release DrugDelivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York,1978.

A Tweak/Tweak-R blocking agent (e.g., an antibody or soluble Tweak-Rprotein) can be modified, e.g., with a moiety that improves itsstabilization and/or retention in circulation, e.g., in blood, serum, orother tissues, e.g., by at least 1.5, 2, 5, 10, or 50 fold. The modifiedblocking agent can be evaluated to assess whether it can reach sites ofdamage after a stroke (e.g., by using a labeled form of the blockingagent).

For example, the Tweak/Tweak-R blocking agent (e.g., an antibody orsoluble Tweak-R protein) can be associated with a polymer, e.g., asubstantially non-antigenic polymer, such as a polyalkylene oxide or apolyethylene oxide. Suitable polymers will vary substantially by weight.Polymers having molecular number average weights ranging from about 200to about 35,000 Daltons (or about 1,000 to about 15,000, and 2,000 toabout 12,500) can be used.

For example, a Tweak or a TweakR binding antibody can be conjugated to awater-soluble polymer, e.g., a hydrophilic polyvinyl polymer, e.g.polyvinylalcohol or polyvinylpyrrolidone. A non-limiting list of suchpolymers include polyalkylene oxide homopolymers such as polyethyleneglycol (PEG) or polypropylene glycols, polyoxyethylenated polyols,copolymers thereof and block copolymers thereof, provided that the watersolubility of the block copolymers is maintained. Additional usefulpolymers include polyoxyalkylenes such as polyoxyethylene,polyoxypropylene, and block copolymers of polyoxyethylene andpolyoxypropylene (Pluronics); polymethacrylates; carbomers; and branchedor unbranched polysaccharides.

When the Tweak/Tweak-R blocking agent (e.g., an antibody or solubleTweak-R protein) is used in combination with a second agent, the twoagents can be formulated separately or together. For example, therespective pharmaceutical compositions can be mixed, e.g., just prior toadministration, and administered together or can be administeredseparately, e.g., at the same or different times.

Administration

The Tweak/Tweak-R blocking agent (e.g., an antibody or soluble Tweak-Rprotein) can be administered to a subject, e.g., a human subject, by avariety of methods. For many applications, the route of administrationis one of: intravenous injection or infusion (IV), subcutaneousinjection (SC), intraperitoneally (IP), or intramuscular injection. Insome cases, administration may be directly into the CNS, e.g.,intrathecal or intracerebroventricular (ICV). The blocking agent can beadministered as a fixed dose, or in a mg/kg dose.

The dose can also be chosen to reduce or avoid production of antibodiesagainst the Tweak/Tweak-R blocking agent.

The route and/or mode of administration of the blocking agent can alsobe tailored for the individual case, e.g., by monitoring the subject,e.g., using assessment criteria discussed herein.

Dosage regimens are adjusted to provide the desired response, e.g., atherapeutic response. For example, doses in the range of 0.1-100 mg/kg,1 mg/kg-100 mg/kg, 0.5-20 mg/kg, 0.1-10 mg/kg or 1-10 mg/kg can beadministered. A particular dose may be administered more than once,e.g., at periodic intervals over a period of time (a course oftreatment). For example, the dose may be administered every 2 months,every 6 weeks, monthly, biweekly, weekly, or daily, as appropriate, overa period of time to encompass at least 2 doses, 3 doses, 5 doses, 10doses, or more.

Dosage unit form or “fixed dose” as used herein refers to physicallydiscrete units suited as unitary dosages for the subjects to be treated;each unit contains a predetermined quantity of active compoundcalculated to produce the desired therapeutic effect in association withthe required pharmaceutical carrier and optionally in association withthe other agent.

Single or multiple dosages may be given. Alternatively, or in addition,the blocking agent may be administered via continuous infusion. Thetreatment can continue for days, weeks, months or even years.

A pharmaceutical composition may include a “therapeutically effectiveamount” of an agent described herein. Such effective amounts can bedetermined based on the effect of the administered agent, or thecombinatorial effect of agents if more than one agent is used. Atherapeutically effective amount of an agent may also vary according tofactors such as the disease state, age, sex, and weight of theindividual, and the ability of the compound to elicit a desired responsein the individual. A therapeutically effective amount is also one inwhich any toxic or detrimental effects of the composition is outweighedby the therapeutically beneficial effects.

Devices and Kits

Pharmaceutical compositions that include the Tweak/Tweak-R blockingagent (e.g., an antibody or soluble Tweak-R) can be administered with amedical device. The device can designed with features such asportability, room temperature storage, and ease of use so that it can beused in emergency situations, e.g., by an untrained subject or byemergency personnel in the field, removed to medical facilities andother medical equipment. The device can include, e.g., one or morehousings for storing pharmaceutical preparations that includeTweak/Tweak-R blocking agent, and can be configured to deliver one ormore unit doses of the blocking agent.

For example, the pharmaceutical composition can be administered with aneedleless hypodermic injection device, such as the devices disclosed inU.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880;4,790,824; or 4,596,556. Examples of well-known implants and modulesinclude: U.S. Pat. No. 4,487,603, which discloses an implantablemicro-infusion pump for dispensing medication at a controlled rate; U.S.Pat. No. 4,486,194, which discloses a therapeutic device foradministering medicants through the skin; U.S. Pat. No. 4,447,233, whichdiscloses a medication infusion pump for delivering medication at aprecise infusion rate; U.S. Pat. No. 4,447,224, which discloses avariable flow implantable infusion apparatus for continuous drugdelivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system. Many otherdevices, implants, delivery systems, and modules are also known.

A Tweak/Tweak-R blocking agent (e.g., an antibody or soluble Tweak-Rprotein) can be provided in a kit. In one embodiment, the kit includes(a) a container that contains a composition that includes a Tweak or aTweak receptor blocking agent, and optionally (b) informationalmaterial. The informational material can be descriptive, instructional,marketing or other material that relates to the methods described hereinand/or the use of the agents for therapeutic benefit. In an embodiment,the kit includes also includes a second agent for treating stroke. Forexample, the kit includes a first container that contains a compositionthat includes the Tweak/Tweak-R blocking agent, and a second containerthat includes the second agent.

The informational material of the kits is not limited in its form. Inone embodiment, the informational material can include information aboutproduction of the compound, molecular weight of the compound,concentration, date of expiration, batch or production site information,and so forth. In one embodiment, the informational material relates tomethods of administering the Tweak/Tweak-R blocking agent (e.g., anantibody or soluble Tweak-R protein), e.g., in a suitable dose, dosageform, or mode of administration (e.g., a dose, dosage form, or mode ofadministration described herein), to treat a subject who has had astroke or who is at risk for stroke. The information can be provided ina variety of formats, include printed text, computer readable material,video recording, or audio recording, or a information that provides alink or address to substantive material.

In addition to the blocking agent, the composition in the kit caninclude other ingredients, such as a solvent or buffer, a stabilizer, ora preservative. The blocking agent can be provided in any form, e.g.,liquid, dried or lyophilized form, preferably substantially pure and/orsterile. When the agents are provided in a liquid solution, the liquidsolution preferably is an aqueous solution. When the agents are providedas a dried form, reconstitution generally is by the addition of asuitable solvent. The solvent, e.g., sterile water or buffer, canoptionally be provided in the kit.

The kit can include one or more containers for the composition orcompositions containing the agents. In some embodiments, the kitcontains separate containers, dividers or compartments for thecomposition and informational material. For example, the composition canbe contained in a bottle, vial, or syringe, and the informationalmaterial can be contained in a plastic sleeve or packet. In otherembodiments, the separate elements of the kit are contained within asingle, undivided container. For example, the composition is containedin a bottle, vial or syringe that has attached thereto the informationalmaterial in the form of a label. In some embodiments, the kit includes aplurality (e.g., a pack) of individual containers, each containing oneor more unit dosage forms (e.g., a dosage form described herein) of theagents. The containers can include a combination unit dosage, e.g., aunit that includes both the Tweak or a Tweak receptor blocking agent andthe second agent, e.g., in a desired ratio. For example, the kitincludes a plurality of syringes, ampules, foil packets, blister packs,or medical devices, e.g., each containing a single combination unitdose. The containers of the kits can be air tight, waterproof (e.g.,impermeable to changes in moisture or evaporation), and/or light-tight.

The kit optionally includes a device suitable for administration of thecomposition, e.g., a syringe or other suitable delivery device. Thedevice can be provided pre-loaded with one or both of the agents or canbe empty, but suitable for loading.

Combination Therapies

The methods and compositions described herein can be used in combinationwith other therapies for inflammatory diseases, such as corticosteroids,NSAIDs, and dialysis.

All references, including patent documents, disclosed herein areincorporated by reference in their entirety.

EXAMPLES Example 1: Tweak is a Biomarker for Lupus Nephritis

Methods

Patients:

Patients for this study were recruited from 2 lupus centers:

1) Ohio State University College of Medicine, Columbus, Ohio, which runsthe Ohio SLE Study (OSS). The OSS is a prospective longitudinalinvestigation of risk factors for SLE flare in recurrently activepatients with renal or non-renal SLE, including patients of Caucasian(70%), African American (29%) and Asian (1%) descent; and

2) the Albert Einstein College of Medicine (AECOM) Lupus Cohort, Bronx,N.Y., which includes the Jacobi and Montefiore Medical Centers lupusclinics. Altogether, lupus clinics at Jacobi and Montefiore MedicalCenters regularly follow around 350 lupus patients, of Caucasian (10%),African American (45%) and Hispanic (45%) descent. The studies at bothcenters have been approved by their respective IRBs.

Both lupus cohorts (OSS and AECOM) follow similar procedures. Firstly,all patients enrolled fulfill at least 4 of the 1982 revised AmericanCollege of Rheumatology criteria for the diagnosis of SLE. The patientsare seen regularly about once every 1-2 months, and at each visit theyare clinically evaluated and receive care from a physician. Routinelaboratory evaluation is usually performed at each visit, including CBC,serum chemistry, ESR, CRP, serum C₃, C₄, anti-nuclear antibodies (ANA)and anti-dsDNA titers, urinalysis, spot urine protein/creatinine ratioand/or a 24 hour urine collection. However, due to different clinicalneeds, a number of the patients included in the cohorts underwent onlypartial laboratory evaluation which did not include all of the abovetests. All available laboratory values were used in the analysis.

At the time of the visit, each patient provides a freshly voided urinespecimen. The fresh AECOM urine samples were frozen in small aliquotswithout further manipulation at −80° C. for later analysis, while thesamples at OSS were first centrifuged to remove sediment before beingfrozen at −80° C. This difference in sample handling did not materiallyaffect the results since results were generally consistent among the 2centers.

Classification of SLE Nephritis Activity Status.

Kidney disease activity was assessed by the renal SLEDAI (rSLEDAI) scorethat consists of the 4 kidney-related items of the SLE Disease ActivityIndex (hematuria, pyuria, proteinuria and urinary casts) (Bombardier etal., The development and validation of the SLE Disease Activity Index(SLEDAI). Arthritis Rheum 1992 vol: 35:630-40). The presence of each oneof the 4 parameters gives a score of 4 points; thus, the rSLEDAI scorecan range from 0 (non-active renal disease) to a maximal score of 16.The patients enrolled in the OSS were additionally classified as to thepresence of active or chronic stable disease, and the severity of renaland non-renal flares, based on criteria described in detail by Rovin etal (Rovin et al., J Am Soc Nephrol 2005; 16:467-73). In brief, thepatients were first divided into a renal disease group (based on akidney biopsy that demonstrated immune complex-mediatedglomerulonephritis, as well as evidence of major renal manifestationspast or present attributable to SLE, such as 24 hr urineprotein/creatinine ratio>1 and/or elevated serum creatinine of over 1.1mg/dl in women and 1.3 mg/dl in men) and a non-renal disease group(normal serum creatinine, urine sediment with <5 red blood cells perhigh-power field and no casts). The patients were further divided withinthe groups as to whether they were undergoing a flare, and as to thenature of that flare (renal or non-renal). Renal flares were classifiedas mild, moderate or severe, based on laboratory tests such as abnormalurine sediment, an elevation of serum creatinine, or a worsening inproteinuria. Non-renal flares were recognized when the patient developedone or more symptoms or signs of non-renal SLE that required themanaging physician to increase therapy. These non-renal flares were alsosorted into mild, moderate or severe based on the severity andlife-threatening potential of the disease manifestations present.

Urinary TWEAK Measurement.

Urinary TWEAK (uTWEAK) levels were determined by ELISA, as follows:microtiter plates were coated with the BEB3 murine monoclonal anti-TWEAKantibody at 2 μg/ml in bicarbonate buffer overnight. The plates werethen blocked by 3% BSA/PBS for 6 hours, washed, and the urine samplesdiluted 1:3 in 3% BSA/PBS were added. In addition, serial dilutions ofrecombinant soluble human TWEAK were added to the plate to construct astandard curve. The plate was then incubated overnight at 4° C., washed,and a solution of pre-mixed biotinylated murine anti-TWEAK antibody P5G9(Campbell et al., J Immunol 2006; 176:1889-98) and avidin-horseradishperoxidase (HRP) (0.5 μg/ml and 1:250 final, respectively) added for onehour at room temperature. The plate was washed, followed by the additionof a developer solution, and the optical density read after 10-20minutes. uTWEAK assays were performed blindly, without knowledge of thepatient's disease status or activity.

Urinary MCP-1 (uMCP-1) and Urokinase Plasminogen Activator Receptor(UPAR) Measurement.

The levels of MCP-1 and uPAR were measured by commercial ELISA kits,according to manufacturer's directions (BioSource International,Camarillo, Calif. and R&D Systems, Minneapolis, Minn., respectively)

Cytokine levels were normalized to urine creatinine concentrationsmeasured in the same spot urine. uTWEAK and uMCP-1 are expressed aspg/mg creatinine (pg/mg Cr), while UPAR levels are expressed as ng/mgCr.

Standardization.

To compare the C3, C4 and anti-dsDNA laboratory measurements obtained atdifferent centers and measured in different laboratories, values werestandardized by dividing the value received from the laboratory for eachpatient by the mid normal range at that same laboratory. For example, ifthe range for serum C4 in a particular laboratory was 16 to 54 and themeasured value was 10, the standardized C4 was calculated as 10 dividedby (16+54)/2=10/35=0.29.

Statistical Analysis.

Data is shown as mean±SEM. Testing among two groups was performed by theMann-Whitney U test. Correlations were determined by the Spearman rankcorrelation coefficient (ρ). P<0.05 was considered significant. Thestatistics program used was GraphPad Prism version 4.03 (GraphPadSoftware, San Diego, Calif.).

Results

The AECOM and OSS cohorts are similar in most aspects, including theage, gender, prevalence of active renal disease, and complement levels.However, significant differences between the 2 cohorts are found in theserum creatinine and proteinuria measurements. This difference may beattributed to several possible factors. While the patients in the OSScohort were recruited from a renal clinic, patients in the AECOM cohortwere recruited from lupus clinics. Furthermore, as described above, oneof the criteria for inclusion in the OSS cohort was a kidney biopsy.Therefore, chronic renal changes, such as elevated serum creatinineand/or proteinuria, may have been more frequent in the OSS than in theAECOM cohort. Nevertheless, as the mean rSLEDAI score in patients withactive LN is not significantly different between the 2 groups, itappears that the OSS and AECOM cohorts are relatively comparable interms of acute inflammatory renal changes.

To determine whether TWEAK correlates with LN activity, we compareduTWEAK levels of patients with active LN (rSLEDAI score≧4) with those ofpatients who never had kidney involvement, or those with previous kidneyinvolvement whose disease was quiescent, i.e. non-active renal disease(n=78). As shown in FIG. 1A, patients with active renal disease (n=43)have higher levels of uTWEAK than lupus patients with non-active ornever renal disease (n=35) (21.57±4.6 versus 10.03±1.4 pg/mg Cr,P=0.001). Moreover, uTWEAK correlated significantly with rSLEDAI scores(ρ=0.405, P<0.001; FIG. 1B). uTWEAK levels displayed significantcorrelation with the total SLEDAI score, performed only in the AECOMcohort (n=30, ρ=0.421, P=0.022); however, this correlation was no longersignificant when only non-renal components of the index were correlatedwith uTWEAK. When uTWEAK levels in SLE patients with chronic, stabledisease (n=20) were compared with those undergoing a flare (n=31),patients with active disease had significantly higher uTWEAK levels thanthose with stable SLE (13.61±1.5 and 9.22±1.7 pg/mg Cr, respectively,P=0.037; FIG. 2A). Furthermore, in the more limited subgroup of lupuspatients with previous renal involvement (n=35), a trend toward higheruTWEAK levels was found in patients undergoing a flare (n=23) as opposedto those with chronic stable renal disease (n=12) (14.54±1.6 and9.39±2.0 pg/mg Cr, respectively, P=0.058; FIG. 2B). Additionally, whenonly patients undergoing a flare were considered separately (n=31),there were significantly higher uTWEAK levels in those patientsundergoing a renal flare (n=23) as opposed to the patients undergoing anon-renal flare (n=8) (14.55±1.6 pg/mg Cr and 8.34±3.0 pg/mg Cr,respectively, P=0.03; FIG. 3). There were no significant differencesbetween uTWEAK levels of patients with varying flare severities(mild-moderate versus severe), or with different WHO classes of LN.

To begin to determine the utility of uTWEAK to help clinically monitorrenal disease activity over time, we analyzed uTWEAK levels in the 6patients that had 2 urine samples available, between which theytransformed from a chronic stable disease state to a flare. As shown inFIG. 4A, in 4 out of the 6 patients there was a marked elevation inuTWEAK levels as the flare was developing. Among these 6 patients, 3 haduTWEAK measurements at 3 different time points. One patient had aparadoxical decrease in uTWEAK levels while undergoing a flare (dottedline in FIG. 4A). The two remaining patients are depicted in FIG. 4B-C,which demonstrate how uTWEAK levels fluctuated in parallel with renalactivity (as measured by rSLEDAI) and the OSS predefined criteria forflare status. Furthermore, as shown in FIG. 4C, uTWEAK levels in thispatient anticipated renal disease activity even better than the rSLEDAIscore. While the patient's renal disease was clinically worsening anduTWEAK levels concomitantly increased, the rSLEDAI score remainedunchanged.

As urinary MCP-1 (uMCP-1) has already been recognized as a biomarker forLN and TWEAK stimulates the production of MCP-1 by mesangial cells andpodocytes, we investigated the correlation between uMCP-1 and uTWEAK,and indeed found a strong correlation (ρ=0.501, P<0.001; FIG. 5A). Inaddition, as shown in FIG. 5B-D, uTWEAK correlated (albeit moderately)with other common serologic indicators of SLE activity such asanti-dsDNA antibodies (ρ=0.459, P=0.008), and complement components C₃and C₄ (ρ=−0.262 and −0.269, respectively, P<0.02). Moreover, uTWEAKcorrelated with systemic inflammatory activity, as measured by theerythrocyte sedimentation rate (ρ=0.373, P=0.013) (data not shown).

We found that uTWEAK did not correlate with the degree of proteinuria(P=0.562). One likely explanation for this observation is that thesource of uTWEAK is the kidneys reflecting local inflammatory activity,rather than resulting from damage to the glomerular filtration barrierand non-specific protein loss into the urine. Additional reinforcementfor the above hypothesis comes from the fact that uTWEAK does notcorrelate with the levels of TWEAK in the serum (data not shown),indicating that uTWEAK is not simply a reflection of serum TWEAKconcentrations. In addition, while urinary levels of the protein UPARdid not correlate with proteinuria (ρ=0.112, P=0.570; n=28), in contrastto uTWEAK, urinary UPAR levels did not correlate with renal diseaseactivity (ρ=−0.024, P=0.891; n=36). This finding supports the conclusionthat the correlation of uTWEAK with disease activity is specific, as notall urinary proteins correlate with renal disease activity or damage.Finally, uTWEAK negatively correlated with plasma BUN (ρ=−0.372,P=0.036), while a similar trend was noted with plasma creatinine(ρ=−0.206, P=0.066).

Example 2: Blocking Tweak Improves Glomerulonephritis

The role of TWEAK/Fn14 interactions in the pathogenesis of lupusnephritis (LN) in SLE is demonstrated.

Methods:

We analyzed the effect of Fn14 deficiency on progression and severity ofLN in the cGVH model of SLE. We chose this murine SLE model, as the Fn14knockout (KO) was already bred into the C57Bl/6 (B6) background, whichis susceptible to disease induction. In this model, a single injectionof 10⁸ MHC II incompatible splenocytes to unirradiated mice inducesautoantibodies and renal disease characteristic of lupus within 2-4weeks. In addition, we analyzed the effect of anti-TWEAK antibodies (Ab)on Fn14 wild type (WT) mice with cGVH induced lupus.

Results:

We used B6.CH2bm12/Kheg (bm12) mice, a B6 derived mouse strain with onlya three amino acid change in the I-Ab chain that is sufficient to inducestrong alloreactivity between B6 and bm12. We compared B6 Fn14 WT and KOmice that were each injected with bm12 donor splenocytes. Control groupsnot expected to develop disease included B6 WT and Fn14 KO mice injectedwith B6 splenocytes. We found that titers of IgG and IgM anti-dsDNA,histone, and chromatin Ab were no different between B6 Fn14 WT and KOmice injected with alloreactive splenocytes. However, kidney disease, asassessed by proteinuria, was significantly less severe in Fn14 KO miceat 6, 8, and 10 weeks. Furthermore, kidney staining for MCP-1 and RANTESwas significantly decreased in Fn14 KO as compared to Fn14 WT mice withinduced lupus. Finally, we found that B6 Fn14 WT mice with induced lupustreated with the P5G9 anti-TWEAK mAb (200 mg×2/week I.P.) hadsignificantly less proteinuria than mice treated with P1.17 (isotypematched control mAb) or PBS.

Conclusion:

Inhibition of TWEAK signaling, by genetically deleting the Fn14 receptoror by anti-TWEAK mAb treatment, significantly improvedglomerulonephritis in the cGVH model of lupus. Systemic autoantibodylevels were not significantly altered.

We claim:
 1. A method of treating lupus nephritis in a human subject inneed thereof, the method comprising: (a) measuring elevated levels ofsoluble TNF-like weak inducer of apoptosis (TWEAK) in a biologicalsample from the human subject in need thereof as compared to a control,wherein the control is a biological sample from a healthy subject, acohort of healthy subjects, or is a baseline value from the humansubject in need thereof and (b) administering a therapeuticallyeffective amount of a TWEAK/TWEAK-R blocking agent to the human subjectin need thereof.
 2. The method of claim 1, wherein the TWEAK/TWEAK-Rblocking agent is selected from the group consisting of an anti-TWEAKantibody or antigen-binding fragment thereof, an extracellular region ofa TWEAK receptor, wherein the extracellular region consists of aminoacids 28-X1 of SEQ ID NO:2, wherein X1 is an amino acid selected fromthe group consisting of amino acids 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, and 80 of SEQ ID NO:2, and an anti-fibroblast growthfactor-inducible 14 (Fn14 blocking antibody or antigen-binding fragmentthereof.
 3. The method of claim 1, wherein the TWEAK/TWEAK-R blockingagent is an anti-TWEAK antibody or antigen-binding fragment thereof oran anti-Fn14 blocking antibody or antigen-binding fragment thereof, andwherein the antibody or antigen-binding fragment thereof is selectedfrom the group consisting of a single chain antibody, Fab fragment,F(ab′)2 fragment, Fd fragment, Fv fragment, and dAb fragment.
 4. Themethod of claim 1, wherein the human subject exhibits decreasedproteinuria as a result of administering the TWEAK/TWEAK-R blockingagent.
 5. The method of claim 1, wherein the biological sample is aurine sample, a serum sample, a plasma sample, or a cerebrospinal fluidsample.
 6. The method of claim 1, wherein the biological sample is aurine sample.
 7. The method of claim 1, wherein soluble TWEAK ismeasured by immunoassay or dipstick immunoassay.
 8. The method of claim1, wherein soluble TWEAK is measured by ELISA or sandwich ELISA.
 9. Themethod of claim 1, wherein the TWEAK/TWEAK-R blocking agent is ananti-TWEAK antibody or antigen-binding fragment thereof.
 10. The methodof claim 1, wherein the TWEAK/TWEAK-R blocking agent is an anti-Fn14blocking antibody or antigen-binding fragment thereof.
 11. The method ofclaim 5, wherein the TWEAK/TWEAK-R blocking agent is an anti-TWEAKantibody or antigen-binding fragment thereof.
 12. The method of claim 5,wherein the TWEAK/TWEAK-R blocking agent is an anti-Fn14 blockingantibody or antigen-binding fragment thereof.
 13. The method of claim 6,wherein the TWEAK/TWEAK-R blocking agent is an anti-TWEAK antibody orantigen-binding fragment thereof.
 14. The method of claim 6, wherein theTWEAK/TWEAK-R blocking agent is an anti-Fn14 blocking antibody orantigen-binding fragment thereof.